Recombinant Mycobacterium Encoding A Heparin-Binding Hemagglutinin (HBHA) Fusion Protein And Uses Thereof

ABSTRACT

Recombinant Mycobacteria (rMyc) which contain sequences encoding a heparin-binding hemagglutinin (HBHA) fission protein are provided, as are methods of making and using the rMyc and the fusion protein. The fusion protein includes an amino terminal mycobacterial antigen Ag85B leader peptide and transcription of the fusion protein is driven by an Ag85B promoter sequence. The recombinant fusion protein is produced in abundance by the rMyc, is post-translationally methylated, and is highly antigenic.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to recombinant Mycobacteria whichcontain and express sequences encoding a heparin-binding hemagglutinin(HBHA) fusion protein. The fusion protein contains an amino terminalmycobacterial antigen Ag85B leader sequence, and transcription of thefusion protein is driven by a suitable promoter, e.g. the Ag85Bpromoter. The invention also provides methods of making and using therecombinant Mycobacteria and the recombinant fusion protein, e.g. asvaccinogens.

Background of the Invention

Tuberculosis (TB) is a global public health problem resulting in 8million new cases and 2 million deaths each year. A particularlyproblematic aspect of TB diagnosis and treatment is the ability of theMycobacterium tuberculosis (Mtb) bacillus to enter a latent,asymptomatic state and to persist in latently infected individuals forlong periods of time. Such individuals are susceptible to reactivationof the disease due to, for example, immune suppression caused bydiseases or conditions such as HIV, treatments such as chemotherapy andthe use of corticosteroids, the waning of immunity that accompaniesaging, etc. An estimated 2 billion persons (one-third of the world'spopulation) are latently infected with Mtb at present, and activation oflatent tuberculosis accounts for most new cases of active disease.Reactivation is associated with inflammation, necrosis and cavitation ofthe lung, a process that results in draining of the lesions into thebronchus. Aerosols generated when individuals with bronchial lesionscough causes dissemination of the Mtb organism to uninfected,susceptible persons, and the transmission cycle is thus maintained.

The only currently available vaccine against TB, Mycobacterium bovis(Bacille Calmette-Guérin) (BCG), was first introduced in 1921. BCG hasbeen widely utilized and while studies show that for some purposes BCGis effective (e.g. against disseminated TB), it is known to beineffective with respect to preventing the development, persistence andreactivation of latent TB.

There is an ongoing need to develop improved, more effective vaccinesagainst TB. In particular, there is a need to develop vaccines thatprovide protection against the development, maintenance and/orreactivation of latent tuberculosis infection.

One protein that has been proposed for use in TB vaccines is theheparin-binding hemagglutinin (HBHA) protein. HBHA is a 22-kDa,methylated, surface-exposed protein that mediates the interaction of thetubercle bacilli with the host, acting as an adhesin for nonphagocyticcells. Methylation of the C-terminal lysine residues is known to affectboth the biochemical and immunological properties of the protein, andseveral experimental findings have implicated HBHA in the process ofextrapulmonary dissemination of Mtb (Pethe et al., 2001. Nature412:190-194.). Temmerman et al. (Nature Medicine 10, 935-941 (2004))showed that covalent methylation of HBHA is necessary for theelicitation of a protective T cell response in mice challenged with Mtb,and Zannetti et al. showed that purified methylated HBHA is stronglyrecognized by sera obtained from TB patients compared to controls,whereas unmethylated HBHA is not (Clin Diagn Lab Immunol September 2005vol. 12 no. 9 1135-1138). In light of these and other studies, it hasbeen proposed that the development of an HBHA-based vaccine mayrepresent an effective strategy to prevent and/or treat TB.

U.S. Pat. No. 7,829,103 to Pethe et al., the complete contents of whichis hereby incorporated by reference in entirety, reports immunogeniccompositions comprising methylated recombinant HBHA. According to Pethc,the HBHA may be produced by one of two methods: either by 1) producingrecombinant non-methylated HBHA protein in a heterologous cell(Escherichia coli or Mycobacterium smegmatis) and thenpost-translationally methylating the purified recombinant HBHA using achemical or enzymatic method; or 2) using a recombinant cell toco-express nucleotide sequences encoding HBHA and a mycobacterialmethyltransferase. Method 1 involves multiple steps for proteinpreparation; method 2 involves the use of a heterologous cell that isnot administrable as a vaccine. Further, the bacterial strains employedby Pethe were antibiotic resistant, and no discussion of optimizingprotein yields is provided. Thus, there remains a need in the art for arecombinant Mtb that is capable of being used as a vaccinogen, and/orfor producing sufficient quantities of HBHA to be clinically relevant,both in vitro and in vivo, and/or for producing large quantities of HBHAin a manufacturing setting for later use in clinical applications.

SUMMARY OF THE INVENTION

The invention provides recombinant Mycobacteria (rMyc) which contain andexpress nucleic acid sequences encoding a heparin-binding hemagglutinin(HBHA) fusion protein. The fusion protein includes a mycobacterialantigen Ag85B leader peptide attached at the amino terminus. The fusionprotein is post-translationally methylated, resulting in a protein witha methylation pattern that is the same as or highly similar to that ofnative HBHA. The resulting fusion protein is thus highly antigenic, andcopious amounts of the highly antigenic fusion protein can be producedusing the methods of the invention. Transcription of the fusion proteinis driven by a suitable promoter, which can be constitutive orinducible. In one embodiment, the promoter is the Ag85B promotersequence, The invention also provides methods of making the rMyc (e.g.on an industrial scale), methods of using the rMyc e.g. as a vaccinogenand/or to elicit an immune response, or to make the fusion protein; orto produce seed cultures; and methods of making and using the fusionprotein e.g. as a vaccinogen and/or to elicit an immune response, or asa diagnostic, for example, to detect latent tuberculosis infections.

It is an object of this invention to provide a recombinant Mycobacteriumthat is genetically engineered to contain and express a nucleic acidfusion sequence encoding an Ag85B leader sequence attached to an aminoterminus of a mycobacterial heparin-binding hemagglutinin (HBHA)protein, the fusion sequence being operably linked to a promoter. Insome embodiments, the Mycobacterium is, for example, Mycobacteriumtuberculaosis, Mycobacterium bovis, or Mycobacterium smegmatis. In otherembodiments, the Mycobacterium is Mycobacterium bovis e.g. Mycobacteriumbovis (Bacille Calmette-Guérin) (BCG). In some embodiments, the BCG is aBCG Danish Statens Serum Institut (SSI) strain and may, for example,express a pfo gene such as a pfo gene from Clostridium perfringens. Insome embodiments, the Mycobacterium is an auxotroph, for example, apantothenic acid auxotroph.

In some embodiments of the invention, the nucleic acid fusion sequenceis

(SEQ ID NO: 1) atgagacgac tttgcgcccg aatcgacatt tggcctccacacacggtatg ttctggcccg agcacacgac gacatacaggacaaaggggc acaagtatgg ccacagacgt gagccgaaagattcgagctt ggggacgccg attgatgatc ggcacggcagcggctgtagt ccttccgggc ctggtggggc ttgccggcggagcggcaacc gcgggcgcgt tctccatggc tgaaaactcgaacattgatg acatcaaggc tccgttgctt gccgcgcttggagcggccga cctggccttg gccactgtca acgagttgatcacgaacctg cgtgagcgtg cggaggagac tcgtacggacacccgcagcc gggtcgagga gagccgtgct cgcctgaccaagctgcagga agatctgccc gagcagctca ccgagctgcgtgagaagttc accgccgagg agctgcgtaa ggccgccgagggctacctcg aggccgcgac tagccggtac aacgagctggtcgagcgcgg tgaggccgct ctagagcggc tgcgcagccagcagagcttc gaggaagtgt cggcgcgcgc cgaaggctacgtggaccagg cggtggagtt gacccaggag gcgttgggtacggtcgcatc gcagacccgc gcggtcggtg agcgtgccgccaagctggtc ggcatcgagc tgcctaagaa ggctgctccggccaagaagg ccgctccggc caagaaggcc gctccggccaagaaggcggc ggccaagaag gcgcccgcga agaaggcggc ggccaagaag gtcacccaga agtag.

In other embodiments, the polypeptide encoded by the nucleic acid fusionsequence has an amino acid sequence: mrrlcaridi wpphtvcsgp strrhtgqrgtsmatdvsrk irawgrrlmi gtaaavvlpg lvglaggaat agafsmaens niddikapllaalgaadlal atvnelitnl reraeetrtd trsrveesra rltklqedlp eqltelrekftaeelrkaae gyleaatsry nelvergeaa lerlrsqqsf eevsaraegy vdqaveltqealgtvasqtr avgeraaklv gielpkkaap akkaapakka apakkaaakk apakkaaakk vtqk(SEQ ID NO: 3).

In some embodiments, the mycobacterial HBHA protein is Mycobacteriumtuberculosis HBHA. In yet other embodiments, the promoter is amycobacterial Ag85B promoter

The invention also provides an isolated recombinant nucleic acidmolecule with a nucleotide sequence:

(SEQ ID NO: 1) atgagacgac tttgcgcccg aatcgacatt tggcctccacacacggtatg ttctggcccg agcacacgac gacatacaggacaaaggggc acaagtatgg ccacagacgt gagccgaaagattcgagctt ggggacgccg attgatgatc ggcacggcagcggctgtagt ccttccgggc ctggtggggc ttgccggcggagcggcaacc gcgggcgcgt tctccatggc tgaaaactcgaacattgatg acatcaaggc tccgttgctt gccgcgcttggagcggccga cctggccttg gccactgtca acgagttgatcacgaacctg cgtgagcgtg cggaggagac tcgtacggacacccgcagcc gggtcgagga gagccgtgct cgcctgaccaagctgcagga agatctgccc gagcagctca ccgagctgcgtgagaagttc accgccgagg agctgcgtaa ggccgccgagggctacctcg aggccgcgac tagccggtac aacgagctggtcgagcgcgg tgaggccgct ctagagcggc tgcgcagccagcagagcttc gaggaagtgt cggcgcgcgc cgaaggctacgtggaccagg cggtggagtt gacccaggag gcgttgggtacggtcgcatc gcagacccgc gcggtcggtg agcgtgccgccaagctggtc ggcatcgagc tgcctaagaa ggctgctccggccaagaagg ccgctccggc caagaaggcc gctccggccaagaaggcggc ggccaagaag gcgcccgcga agaaggcggc ggccaagaag gtcacccaga agtag.

The invention also provides a recombinant fusion protein which comprisesan Ag85B leader sequence covalently attached to an amino terminus of amycobacterial heparin-binding hemagglutinin (HBHA) protein. In oneembodiment, the entire fusion protein is transcribed as one mRNA andtranslated as a single polypeptide (protein). In one embodiment, therecombinant fusion protein has an amino acid sequence: mrrlcaridiwpphtvcsgp strrhtgqrg tsmatdvsrk irawgrrlmi gtaaavvlpg lvglaggaatagafsmaens niddikapll aalgaadlal atvnelitnl reracetrtd trsrveesrarltklqedlp eqltelrekf taeclrkaae gyleaatsry nelvergeaa lerlrsqqsfccvsaraegy vdqavcltqec algtvasqtr avgeraaklv gielpkkaap akkaapakkaapakkaaakk apakkaaakk vtqk (SEQ ID NO: 3). In some embodiments, therecombinant fusion protein is methylated.

The invention also provides methods of producing a recombinant fusionprotein that comprises an Ag85B leader sequence attached to an aminoterminus of a mycobacterial heparin-binding hemagglutinin (HBHA)protein. The method comprises: 1) transfecting a bacterial cell such asa Mycobacterium cell with a nucleic acid sequence encoding therecombinant fusion protein; 2) growing the transfected bacterium (e.g. aMycobacterium) cell under conditions which allow the bacterium cell toproduce the recombinant fusion protein; and 3) obtaining the recombinantfusion protein. In some embodiments, the transfecting is carried out byelectroporation.

The invention also provides methods of determining whether a subject hasa latent tuberculosis infection. The method comprises determining thepresence or absence of immune reactivity of the patient to a recombinantfusion protein comprising an Ag85B leader sequence attached to an aminoterminus of a mycobacterial heparin-binding hemagglutinin (HBHA)protein. The presence of immune reactivity indicates that the subjecthas a latent tuberculosis infection. Determining the presence or absenceof immune reactivity may include, for example, 1) obtaining a biologicalsample from the subject, and 2) detecting the presence or absence ofimmune reactivity in the biological sample. Exemplary biological samplesinclude but are not limited to sputum samples and serum samples. Inother embodiments, determining the presence or absence of immunereactivity may include the step of detecting includes 1) intradermallyinjecting the recombinant fusion protein into the subject, and 2)determining the presence or absence of immune reactivity at the site ofintradermal injection.

The invention also provides methods of eliciting an immune responseagainst Mycobacterium tuberculosis in a subject in need thereof. Thismethod comprises the step of administering to the subject an amount of arecombinant fusion protein comprising an Ag85B leader sequence attachedto an amino terminus of a mycobacterial heparin-binding hemagglutinin(HBHA) protein sufficient to elicit an immune response in said subject,and may be referred to as a “therapeutic” amount. In one embodiment, theimmune response that is elicited is production of one or more of Bcells, antibodies and T cells. In some embodiments, the immune responseis a protective immune response.

The invention also provides methods of eliciting an immune responseagainst Mycobacterium tuberculosis in a subject in need thereof. Themethod comprises administering to the subject a recombinantMycobacterium comprising a nucleic acid fusion sequence encoding anAg85B leader sequence attached to an amino terminus of a mycobacterialheparin-binding hemagglutinin (HBHA) protein. The fusion sequence isoperably linked to a promoter, and the recombinant Mycobacterium isadministered in an amount sufficient to elicit an immune response in thesubject. In one embodiment, the immune response is production ofantibodies. In some embodiments, the immune response is a protectiveimmune response.

The invention also provides methods of producing recombinantheparin-binding hemagglutinin (rHBHA) protein. The methods comprise 1)growing a culture of recombinant Mycobacteria comprising a nucleic acidfusion sequence encoding an Ag85B leader sequence attached to an aminoterminus of a mycobacterial heparin-binding hemagglutinin (HBHA)protein, wherein said fusion sequence is operably linked to a promoter,under conditions in which the rHBHA is produced. In some embodiments,the method further comprises obtaining the rHBHA protein from theculture. The method may also include purifying the rHBHA protein, e.g.after the step of obtaining the rHBHA. Purifying may be carried outusing one or more physico-chemical technologies such as, for example,chromatography (e.g. one or more of affinity, size exclusion, ionexchange, or hydrophobic interaction chromatography); and/or celldisruption techniques (e.g. one or more of high pressure celldisruption, bead beaters, homogenization, sonication, centrifugation,and the like). The method may also include verifying the identity of therHBHA protein. In some embodiments, the step of growing is carried outin culture by shaking or by fermentation. In some embodiments, growingis carried out using a hatch or a continuous culture.

The invention further provides seed lots of recombinant Mycobacterium.In some embodiments, a seed lot comprises 1) recombinant Mycobacteriacomprising a nucleic acid fusion sequence encoding an Ag85B leadersequence attached to an amino terminus of a mycobacterialheparin-binding hemagglutinin (HBHA) protein, wherein the fusionsequence is operably linked to a promoter; and 2) medium suitable formaintaining the recombinant Mycobacteria in a viable state duringstorage of the seed lot.

The invention also provides methods of preparing a compositioncomprising a heparin-binding hemagglutinin (HBHA) protein. In someembodiments, the method comprises 1) growing a culture of recombinantMycobacteria comprising a nucleic acid fusion sequence encoding an Ag85Bleader sequence attached to an amino terminus of a mycobacterialheparin-binding hemagglutinin (HBHA) protein, wherein the fusionsequence is operably linked to a promoter; 2) obtaining the rHBHAprotein from the culture; 3) purifying the rHBHA protein; and 4)combining purified rHBHA protein with a physiologically acceptablecarrier. The growing may be carried out in shake culture or byfermentation. In addition, method may further comprise adding one ormore additional therapeutically useful agents, including but not limitedto, one or more antigens that are not HBHA, one or more adjuvants, andone or more immunogenicity enhancers, to the rHBHA and physiologicallyacceptable carrier. These compositions may be used, for example, as avaccine, a therapeutic or a diagnostic, or for any other purpose.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the DNA sequence (SEQ ID NO:1) encoding the HBHA fusionprotein. The Ag85B leader peptide is encoded by the underlinednucleotides. FIG. 1B shows the DNA sequence encoding the HBHA fusionprotein as in FIG. 1A but also shows the exemplary Ag85B promotersequence in bold (SEQ ID NO: 2).

FIG. 2 shows the in silico cloning and complementation strategy.

FIG. 3A shows the schematic of the pantothenate complementation plasmid(pKAMCB2) and FIG. 3B shows the cloning of HBHA gene into pKAMCB2.

FIG. 4 shows the schematic unmarking of the pKAMCB2+HBHA clone forkanamycin resistance.

FIG. 5A shows the amino acid sequence of recombinant HBHA (SEQ ID NO: 3)compared with FIG. 5B, which shows the native HBHA from which aminoterminal methionine has been cleaved (SEQ ID NO: 4) and FIG. 5C, whichshows the schematic position of the Ag85B leader at the N-terminus.

FIG. 6A shows the recombinant BCG colonies PCR screened for the HBHAgene. FIG. 6B shows the presence of plasmid backbone and FIG. 6C showsthe absence of the kanamycin resistance marker. Clone no. 5 testedpositive for the presence of HBHA gene, intact plasmid backbone and theabsence of kanamycin gene.

FIG. 7 shows the growth kinetics of AERAS 445, a pantothenate auxotrophof a BCG Danish SSI strain expression a Clostridium perfringens pfogene, and further modified to encode the HBHA fusion protein of theinvention.

FIG. 8 shows the Western blot over expression of HBHA in AERAS-445 using4057D2 anti-HBHA monoclonal antibody. Arrows 1 and 2 indicate endogenousHBHA and recombinant HBHA proteins.

FIG. 9 shows the Western blot over expression of HBHA in AERAS-445 using1G10 antibody. Lanes: 1 AERAS-401; 2 AERAS-413; 3 AERAS 445. Arrows 1and 2 indicate native HBHA and recombinant HBHA proteins.

FIG. 10 shows the Western blot HBHA expression using 1G10 antibody indifferent stages of manufacturing AERAS-445. Arrows 1 and 2 indicatenative HBHA and recombinant HBHA proteins. Lanes: 1 AERAS 413; 2 Stage 2manufacturing sample; 3 Accession Fermentor sample. Arrows 1 and 2indicate endogenous HBHA and recombinant HBHA proteins.

FIGS. 11A and 11B show the yields of HBHA protein in strains determinedby sandwich ELISA; FIG. 11A, depicted graphically, and FIG. 11B,depicted as a bar graph.

FIG. 12 shows the mass spectrometry analysis of rHBHA purified fromAERAS 445.

FIG. 13 shows the mass-spectrometry analysis of the C-terminal end ofrHBHA purified from AERAS 445.

FIGS. 14A and 14B show the antigenicity analysis by ELISA usingmonoclonal antibodies 3921E4 (FIG. 14A): and 4057D2 (FIG. 14B).

DETAILED DESCRIPTION

In one embodiment, the invention provides recombinant Mycobacteria(rMyc) which contain and express nucleic acid sequences encoding aheparin-binding hemagglutinin (HBHA) fusion protein. The recombinantMycobacteria can be administered in vaccine preparations since they arenot antibiotic resistant and are attenuated. The fusion proteinincludes, attached to its amino terminus, a mycobacterial antigen Ag85Bleader peptide sequence. The rMycs of the invention produce largeamounts of the fusion protein, with transcription being driven by asuitable promoter. The fusion protein may also be recovered from therMyc in an industrial manufacturing process and be used as part of avaccine preparation or diagnostic.

In one embodiment, the nucleic acid sequence that encodes the antigenicrecombinant fusion protein of the invention is the DNA sequence depictedin FIG. 1A (SEQ ID NO: 1) As can be seen, the nucleic acid encodes theAg85B leader sequence at nucleotides 1 to 225 (underlined) and the HBHAprotein at nucleotides 226 to 825. In addition, in the exemplaryembodiment depicted in FIG. 1B, at its 5′ end, the nucleic acid containsthe Ag85B promoter sequence at nucleotides 1 to 184 (shown in bold). Inthe embodiment illustrated in FIG. 1B, the translated fusion protein perse that is produced by the cell is thus encoded by nucleotides 185 to1009, i.e. the promoter region is not translated.

In one embodiment, the invention encompasses a nucleic acid with asequence which is or which includes the sequence as set forth in SEQ IDNO: 1 (see FIG. 1A). In another embodiment, the invention encompasses anucleic acid with a sequence that is or includes the sequence as setforth in SEQ ID NO: 2 (sec FIG. 1B). In another embodiment, theinvention encompasses a nucleic acid with a sequence that is or includesthe sequence as set forth in SEQ ID NO: 3 (see below). The inventionalso encompasses DNA that is complementary to SEQ ID NOS: 1 and 2, andalso mRNA that is translated from SEQ ID NOS: 1 and 2 (or complementsthereof), or cDNA based on such mRNA (as well as various DNA-RNA hybridsof these), and encompasses both single and double stranded nucleicacids. Further, those of skill in the art will recognize that, in orderto produce an antigenic recombinant fusion protein as described herein(i.e. comprising an Ag85B leader sequence attached to an amino terminusof a mycobacterial HBHA protein), the precise sequence of SEQ ID NOS: 1and 2 need not be employed. For example, sequences with at least about75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or about 99% homology toSEQ ID NOS: 1 and 2 may also be employed, so long as the translatedpolypeptide is able to be methylated and is sufficiently antigenic toelicit an immune response in a subject to whom it is administered. Theselevels of homology are also applicable to the correspondingcomplementary DNA, RNA, etc. described above. Those of skill in the artare familiar with automated programs or software for determininghomology levels. In addition, those of skill in the art will recognizethat the nucleic acid sequence that encodes the fusion protein may alsoinclude various helpful sequences, e.g. restriction sites for ease ofgenetic manipulation of the sequence. The % homologies described hereincan be determined, for example, using the Smith-Waterman homology searchalgorithm as implemented in MSPRCH program (Oxford Molecular) usinganaffine gap search with the following search parameters: gap openpenalty of 12, and gap extension penalty of 1.

Exemplary variations of the recombinant sequence include but are notlimited to: the substitution of codons which encode conservative aminoacid replacements of some encoded residues; the insertion of e.g.sequences encoding linker or spacer sequences, e.g. between the promoterand the leader sequence, or between the leader sequence and the HBHAencoding sequence; various changes to the sequence to facilitatehandling or manipulation of the sequence, e.g. the insertion of orchange in restriction enzyme sites which flank the sequence; portions ofa vector (e.g. 5′ or 3′ overhangs or sequences complementary to thesame), sequences which encode various tags as described below, etc. Thisis illustrated in the sequence of SEQ ID NO: 3, where additionalnon-coding sequences at the 5′ and 3′ ends of the sequence are shown initalics, and the bold and underlined sequences represent promoter regionand a leader peptide, as described above.

(SEQ ID NO: 12) ACTGTTAATTAA GTGGTCTTCGTCGGCTTGCTTCGAGCGAGCCTACGCGGTGAACGCAAGTTCGGCCTCCCTGGGGGAGCACAGCCGGTAGCCCCGGG CCGCGATTCTGAGAAATCCGCGATAGATCCATACCGCCATACCGTTTGTGAGCCCCCTAAGCACACTTGCTCTGTCCGCGGCGGTAACCGATACGGAAATGAGACGACTTTGCGCCCGAATCGACATTTGGCCTCCACACACGGTATGTTCTGGCCCGAGCACACGACGACATACAGGACAAAGGGGCACAAGTATGGCCACAGACGTGAGCCGAAAGATTCGAGCTTGGGGACGCCGATTGATGATCGGCACGGCAGCGGCTGTAGTCCTTCCGGGCCTGGTGGGGCTTGCCGGCGGAGCGGCAACCGCGGGCGCGTTCTCCATGGCTGAAAACTCGAACATTGATGACATCAAGGCTCCGTTGCTTGCCGCGCTTGGAGCGGCCGACCTGGCCTTGGCCACTGTCAACGAGTTGATCACGAACCTGCGTGAGCGTGCGGAGGAGACTCGTACGGACACCCGCAGCCGGGTCGAGGAGAGCCGTGCTCGCCTGACCAAGCTGCAGGAAGATCTGCCCGAGCAGCTCACCGAGCTGCGTGAGAAGTTCACCGCCGAGGAGCTGCTGTAAGGCCGCCGAGGGCTACCTCGAGGCCGCGACTAGCCGGTACAACGAGCTGGTCGAGCGCGGTGAGGCCGCTCTAGAGCGGCTGCGCAGCCAGCAGAGCTTCGAGGAAGTGTCGGCGCGCGCCGAAGGCTACGTGGACCAGGCGGTGGAGTTGACCCAGGAGGCGTTGGGTACGGTCGCATCGCAGACCCGCGCGGTCGGTGAGCGTGCCGCCAAGCTGGTCGGCATCGAGCTGCCTAAGAAGGCTGCTCCGGCCAAGAAGGCCGCTCCGGCCAAGAAGGCCGCTCCGGCCAAGAAGGCGGCGGCCAAGAAGGCGCCCGCGAAGAAGGCGGCGGCCAAGAAGGTCACCCAGAAGTAGACTAGTT CAT.

Expression of the fusion protein is driven by a promoter sequence thatis operably linked to SEQ ID NO: 1. By “operably linked” it is meantthat the promoter sequence and SEQ ID NO: 1 are arranged within anucleic acid molecule such that expression of SEQ ID NO: 1 is driven orcontrolled by the promoter. In some embodiments, the promoter maydirectly precede SEQ ID NO: 1 in the molecule. In other embodiments,some additional sequences may intervene. In addition, other controlelements that aid in expression of SEQ ID NO: 1 may also be included inthe nucleic acid molecule, e.g. various enhancer sequences, etc.Exemplary promoters that may be used in the practice of the inventioninclude but are not limited to, for example, promoters of genes hsp60,hspX, pBlaF or mtrA, etc. In one embodiment, the promoter is the Ag85Bpromoter, and is arranged with respect to SEQ ID NO: 1 as is depicted inFIG. 1B (the sequence in bold), i.e. is placed directly upstream of SEQID NO: 1.

The fusion protein of the invention, as translated, is an antigenicrecombinant fusion or chimeric protein (polypeptide) which comprises: 1)a mycobacterial HBHA protein sequence, or a functional portion thereof;and 2) an Ag85B leader peptide (or functional portion thereof) attachedto or associated with the amino terminus of the HBHA protein. By “amycobacterial HBHA protein sequence or functional portion thereof” wemean an HBHA protein with a sequence as depicted in FIG. 5B (SEQ ID NO:4) or peptide or polypeptide fragments thereof which are antigenic, i.e.which elicit the production of antibodies which bind to native HBHA,when administered as a component of the fusion protein of the invention.Such fragments may also be sufficient to interact with and bind toheparin. For example, antigenic peptide fragments of about 50 aminoacids or less in length which are comprised within about the last 30 to50 amino acids located at the carboxyl terminus of SEQ ID NO: 4 may beemployed. Peptides or polypeptides which comprise such peptide fragmentsmay also be employed, with a polypeptide being greater than about 50amino acids in length, but generally shorter than a full length HBHAprotein. In some embodiments, such active peptide fragments may be fromabout 10 to about 20 amino acids in length. In other embodiments, thepeptides/polypeptides may comprise or may be the 39-amino acid peptideshown below in SEQ ID NO: 5, or sequences with at least about 90% orgreater (e.g. 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identity to SEQ IDNO: 5. Further description of HBHA proteins and fragments thereof thatmay be used in the practice of the present invention is provided inissued U.S. Pat. No. 6,949,345 (Menozzi et al.), the complete contentsof which is hereby incorporated by reference in entirety.

In some embodiments, the Ag85B leader sequence is attached directly tothe amino terminus of the HBHA protein by virtue of the two having beentranslated as a single polypeptide, from tandem nucleic acid sequenceswithin a nucleic acid molecule. In this case, the attachment is covalentand there is no intervening amino acid sequence between the leadersequence and the HBHA sequence. However, in some embodiments, relativelyshort (e.g. from about 1 to about 10) amino acid linker or spacersequences may be present between the two, e.g. spacers comprisingrelatively small uncharged amino acids such as glycine, alanine, etc. Inaddition, in some embodiments, the fusion protein of the invention mayhave various other modifications, such as the attachment of taggingsequences e.g. to facilitate isolation or detection, e.g. affinity tagssuch as His tags, Isopeptag, glutathiono-S-transfcrasc (GST), chitinbinding protein (CBP), maltose binding protein (MBP), etc.;solubilzation tags such as thioredoxin, MBP, GST, etc.; chromatographytags such as FLAG-tag; epitope tags such as V5-tag, c-myc-tag, HA-tag,etc.; and fluorescent tags such as various green fluorescent protein(GFP) tags and derivatives thereof, etc.

The immunogenic recombinant fusion protein sequence of the presentinvention is methylated, e.g. at the heparin-binding region of the HBHA.In particular, the methyl groups are carried by lysine residues presentin said heparin-binding region. The sequence for said carboxy-terminalregion is as follows:

(SEQ ID NO: 5) KKAAPAKKAAPAKKAAPAKKAAAKKAPAKKAAAKKVTQKThe methyl groups are carried by all or only part of the lysine residuespresent in the C-terminal region of HBHA. Advantageously, at least aboutten lysine residues (e.g. about 10, 11, 12, 13, 14 or 15) out of thefifteen present in the C-terminal region are methylated, with themethylated lysine residues being mono- or di-methylated.

In alternative embodiments, the fusion protein of the invention may besynthesized chemically, e.g. using methodology that is well known in theart. In this embodiment, methylation is or may be carried out in vitro,e.g. as described by above-cited Pethe.

In one embodiment of the invention, the amino acid sequence of therecombinant fusion protein is: mrrlcaridi wpphtvcsgp strrhtgqrgtsmatdvsrk irawgrrlmi gtaaavvlpg Ivglaggaat agafsmaens niddikapllaalgaadlal atvnelitnl reraeetrtd trsrveesra rltklqedlp eqltelrekftaeclrkaae gyleaatsry nelvergeaa lerlrsqqsf eevsaraegy vdqaveltqealgtvasqtr avgeraaklv gielpkkaap akkaapakka apakkaaakk apakkaaakk vtqk(SEQ ID NO: 3). In other embodiments, the amino acid sequence of therecombinant fusion protein is a sequence that is at least about 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to that of SEQID NO: 3, while still retaining sufficient functionality i.e.antigenicity to elicit an immune response in a subject to, whom it isadministered, or still being capable of reacting in assays to detect,e.g. the presence of a latent TB infection in a patient. For example,conservative amino acid substitutions may be made in the sequence,whereby amino acids with positively charged side chains are substitutedby other amino acids with positively charged side chains (e.g. lysine,histidine, arginine); or whereby amino acids with negatively chargedside chains are substituted by other amino acids with negatively chargedside chains (e.g. aspartic acid and glutamic acid); or whereby aminoacids with hydrophobic side chains are substituted by other amino acidswith hydrophobic side chains (alanine, leucine, etc.); or whereby aminoacids with polar uncharged side chains are substituted by other aminoacids with polar uncharged side chains (serine, threonine, etc.); andother substitutions that do not negatively impact the antigenicity ofthe protein. Further, certain other mutations to of deletions of oradditions of single amino acids or short sequences of amino acids (e.g.about 2-5) in the sequence may be tolerated without vitiating theproduction and antigenicity of the fusion protein. Generally, suchchanges are not carried out in the heparin binding region of theprotein, or at least such changes do not perturb the lysine residueswhich are methylated to yield the antigenic form of the protein.

The HBHA protein whose primary amino acid sequence is set forth in SEQID NO: 3 was identified in and derived from M. tuberculosis and is anative Mtb sequence. However, those of skill in the art will recognizethat other HBHA proteins may also be utilized in the practice of theinvention. Generally, the HBHA is identified in or derived from (i.e. isnative to) a mycobacterial species or strain, for example, variousstrains of M. bovis or M. tuberculosis, but may come from any source solong as it functions as described herein, i.e. advantageously largequantities of the protein may be produced as described herein (e.g. atleast about 10 to about 300 or more (e.g. from about 15 to about 250, orfrom about 20 to about 200, or from about 15 to about 150, or from about10 to about 100 μg HBHA per mg of total protein, with the range beinggenerally from about 30 to about 100 g HBHA per mg of total protein).Regardless of the precise quantity, the antigenic quality of the proteinis maintained, i.e. the antigenicity of the protein is comparable tothat of the fusion protein represented by SEQ ID NO: 3, in terms ofeliciting an immune response.

Methods of growing bacterial cultures in order to produce protein arewell known in the art, as are methods of obtaining, and isolating orpurifying proteins produced in this manner. The invention encompassesmethods of making the fusion protein described herein by transfecting(e.g. by electroporation) a suitable mycobacterial cell, growing thetransfected cell under culture conditions which are suitable for thegrowth of the organism and production of the protein by the organism,and then obtaining and purifying the protein. Exemplary conditions andtechniques are described in the Examples section below.

In some embodiments, the nucleic acid that is introduced into themycobacterial cell is contained within a vector such as a plasmid.However, other transfectable or transferable vectors may be used in thepractice of the invention, e.g. various viral vectors, other episomalelements, etc. In addition, in some embodiments, the nucleic acidssequences of interest may be incorporated into the genome of themycobacterium.

The fusion protein that is produced and used in the practice of thepresent invention is, in some embodiments, substantially purified, e.g.a preparation of the protein is generally (e.g. at least 80, 90, 95, oreven 99% or more) free of other proteins, as well as being free of othercellular components, e.g. nucleic acids, lipids, and othermacromolecules. The rMyc of the invention may be employed as a biosourcefor rHBHA production and the rHBHA so-produced may be used for anypurpose. In one embodiment, the purpose is to produce therapeutic immuneresponse stimulating formulations of isolated, substantially purifiedfusion protein, which are discussed below.

The bacterial cells that are used in the practice of the invention maybe any that fulfill the criteria of being suitable for use in a vaccinepreparation. e.g. they are attenuated (i.e. decreased in virulence ordisease causing capacity; rendered innocuous or incapable of causingsymptoms—or causing only minor symptoms—of disease, as is understood inthe art); of not displaying antibiotic resistance; and being antigenic.In some embodiments, the cells are various species or variants ofmycobacteria, (e.g. mutant or recombinant forms) of Mycobacteriumtuberculosis, Mycotabcterium bovis, Mycobacterium smegmatis, or othermycobacteria, etc. In some embodiments, the bacterial cell isMycotabcterium bovis BCG and/or various strains thereof, for example,mutant BCGs selected for a particular property, or recombinant BCGs thathave been genetically manipulated. Exemplary recombinant BCGs aredescribed, for example, in U.S. Pat. Nos. 7,625,572; 7,666,656;7,829,104; and 8,043,857; all to Sun et al., the complete contents ofeach of which are hereby incorporated by reference. For example, the BCGmay be genetically manipulated to contain and express an endosomolyticprotein that is active at neutral pH (e.g. Perfringolysin O fromClostridium perfringens), and may also be auxotrophic and not antibioticresistant, e.g. auxotrophic for the production of leucine, pantothenate,etc., or double auxotrophs such as leucine-pantothenate auxotrophs, etc.In some embodiments, the mycobacterial cell expresses a pfo gene.Exemplary pfo genes include but are not limited to those of bacteriasuch as Clostridium perfringens. However, those of skill in the art willrecognize that other functionally similar proteins exist which couldalso be employed in the practice of the invention, either in their wildtype form, or after being genetically modified to render them suitable.Examples of such endosomalytic proteins include but are not limited toListeriolysin (Llo, produced by Listeria monocytogenes), Pneumolysin(produced by Streptococcus pneumoniae), Streptolysin O (produced byStreptococcus pyogenes), Cerolysin (produced by Bacilus cereus),α-hemolysin (produced by Staphylococcus aureus), etc. In one embodiment,the cell that is employed is a pantothenate auxotroph of a BCG DanishSSI strain expressing a Clostridium perfringens pfo gene, referred toherein as “AERAS-413”. However, those of skill in the art will recognizethat other mycobactieral strains, e.g. other BCG strains such as BCG SSIwithout the pfo gene, Tokyo, Tice, etc., may also be employed as maythose listed in, for example, U.S. Pat. No. 7,666,656, the completecontents of which is hereby incorporated by reference in entirety.

The antigenically active form of the fusion protein of the invention ismethylated. Generally, methylation is carried out by and within the rMyccell, which, in some embodiments, has a native, intrinsic capacity toproduce active methyltransferase enzymes. However, in some embodiments,the cell may be further modified to include nucleic acid sequencesexpressing additional methyltransferases, or further modified to producehigher levels of native methyltransferases.

The present invention also provides compositions for use in eliciting animmune response against M. tuberculosis and/or vaccinating an individualagainst tuberculosis. In one embodiment, the compositions include, as anactive agent, one or more rMyc species or strains, and apharmacologically suitable carrier. In another embodiment, thecompositions include, as an active agent, one or more substantiallypurified fusion proteins as described herein, and a pharmacologicallysuitable carrier. The preparation of such compositions for use asvaccines is well known to those of skill in the art. Typically, suchcompositions are prepared either as liquid solutions or suspensions,however solid forms such as tablets, pills, powders and the like arealso contemplated. Solid forms suitable for solution in, or suspensionin, liquids prior to administration may also be prepared. Thepreparation may also be emulsified. The active ingredients may be mixedwith excipients which are pharmaceutically acceptable and compatiblewith the active ingredients. Suitable excipients are, for example,water, saline, dextrose, glycerol, ethanol and the like, or combinationsthereof. In addition, the composition may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents, and the like. In addition, the composition may contain variousadjuvants (compounds that can induce or increase the humoral and/orcellular immune response towards an antigen or immunogen). If it isdesired to administer an oral form of the composition, variousthickeners, flavorings, diluents, emulsifiers, dispersing aids orbinders and the like may be added. The composition of the presentinvention may contain any such additional ingredients so as to providethe composition in a form suitable for administration. The final amountof active agent in the formulations may vary. However, in general, theamount in the formulations will be from about 1-99%. Advantageously, animmunogenic composition of the invention comprises, per dose, from about0.1 to about 50 μg of fusion protein, and generally from about 1 to 50μg, e.g. about 15 μg of purified HBHA fusion protein. If the compositionof the product comprises a live bacteria (e.g. an attenuated bacteria),the amount of bacteria per dose is generally in the range of from about1×10 to about 1×10′, and is usually about 1×10⁶.

The compositions (preparations, formulations, etc.) of the invention maybe administered by any of the many suitable means which are well knownto those of skill in the art, including but not limited to by injection,intradermally, inhalation, orally, intravaginally, intranasally, byingestion of a food or probiotic product containing the active agent,topically, as eye drops, via sprays, etc. In some embodiments, the modeof administration is by injection or intradermally. In addition, thecompositions may be administered in conjunction with other treatmentmodalities such as substances that boost the immune system, variouschemotherapeutic agents, other antigenic agents, various adjuvants, andthe like.

The invention also provides methods for eliciting an immune response ina subject in need thereof. The method involves, in one embodiment,administering a composition comprising the fusion protein of theinvention, and in another embodiment, administering the rMyc of theinvention. The immune response that is elicited may be one or both ofinnate and adaptive, and may involve both cell-mediated and humoralresponses, for example, the production of antibodies, and/or a B celland/or T cell response, etc. The response may be protective in that arecipient of the vaccine is protected against subsequent challenge withMtb, thereby preventing the development of a TB infection, either activeor latent. Further, if the compositions of the invention areadministered after TB infection has occurred, the immune response may besuch that a latent infection does not result, or, if a latent infectionis already present, reactivation of the latent infection to activedisease may be prevented, or the extent or severity of infection may belessened.

The invention also provides diagnostic tests which utilize the fusionprotein of the invention. In particular, the tests are useful foridentifying individuals who have a latent tuberculosis infection. Thetests typically involve exposure of the immune cells or products ofimmune cells of a subject identified as possibly having latent TB (e.g.due to exposure to the disease, or for some other reason) to the proteinand determining whether or not the immune cells or products thereofreact to or recognize the fusion protein. In one embodiment, exposure iscarried out by obtaining a biological sample from the subject (e.g.saliva, sputum, blood, plasma, etc.) and exposing the sample to thefusion protein in order to detect, e.g. the presence of anti-HBHAantibodies in the sample. In another embodiment, the fusion protein isinjected intradermally (e.g. in the manner of the Mantoux or othersimilar tests) and the reaction of the skin to the protein is observed.

In both embodiments, a positive reaction to the test indicates orconfirms that the subject may or is likely to have a latent tuberculosisinfection.

In some embodiments the invention provides methods for producingcultures of the rMyc of the invention for use as seed lots. Inparticular, methods involving the batch production, purification andtesting of the rMyc are encompassed. Such methods generally involvestepping up production of an initial clonal isolate, growing the isolateon a modest scale in media that is free of serum and animal originprotein, and then inoculating a culture of a desired larger volume(repeating this step as necessary until a sufficient volume isattained); and growing the rMyc culture under conditions that allow therMyc to grow and undergo cell division. Generally, the rMyc is grown toa density (A₆₀₀) of from about 4 to about 6, e.g. about 5. Aliquots ofthe culture are prepared e.g. for commercial purposes, either directlyfrom the culture, or by concentrating the rMyc (e.g. by centrifugation)and resuspending the pellet in a suitable medium. The final amount ofbacteria in a seed lot culture is generally on the order of from about4×10⁷ to about 6×10⁷. Those of skill in the art are familiar withtechniques for the long-term maintenance of such cultures, e.g.cryopreservation, lyophilization, etc. Prior to final preparation andstorage of the rMyc cultures, the rMyc identity and purity may betested/confirmed by methods known to those of skill in the art, e.g. bysampling the culture and analyzing it for the presence of the nucleicacid which encodes the fusion protein, or by analyzing it for thepresence of the fusion protein itself.

In other embodiments, the invention provides methods for producing arecombinant heparin-binding hemagglutinin (rHBHA) protein product. Themethods comprise the steps of a) growing a culture of recombinantMycobacteria comprising a nucleic acid fusion sequence encoding an Ag85Bleader sequence attached to an amino terminus of a mycobacterialheparin-binding hemagglutinin (HBHA) protein, the fusion sequence beingoperably linked to a promoter, and obtaining the rHBHA protein from theculture. The Mycobacteria may have been genetically engineered tocontain and express the nucleic acid fusion sequence. Growth of theculture may be carried out in any suitable manner, e.g. in shakeculture, by fermentation, etc., and the culture may be, for example, abatch or continuous culture.

The production methods typically also include a step of purifying therHBHA protein. Purification of the protein may be carried out by any ofthe many suitable technologies that are known to those of skill in theart. Typically, the technology involves the use of one or morephysico-chemical purification techniques. Exemplary techniques includebut are not limited to: chromatography techniques such as ion exchangechromatography, affinity chromatography, size exclusion chromatography,hydrophobic interaction chromatography, etc.; as well as various celldisruption techniques such as high pressure cell disruption, the use ofbead beaters, homogenization, sonication, centrifugation, etc.Combinations of these and other techniques may also be employed.

The methods may also include a step of verifying the identity of therHBHA protein. Those of skill in the art are familiar with suitabletechniques for verifying the identity and/or purity of proteins andpolypeptides. Exemplary techniques include but are not limited to:electrophoresis, chromatography, mass spectrometry, amino acidsequencing, etc.

The methods may also include a step of combining purified rHBHA proteinwith a physiologically acceptable carrier. Suitable physiologicallyacceptable or compatible carriers are described elsewhere herein. Insome embodiments, the formulated HBHA protein product further comprisesone or more other active agents. For example, one or more antigens thatare not HBHA (i.e. a non-HBHA antigen) may be included. Exemplaryadditional antigens include but are not limited to, for example, thosewhich elicit an immune response against diseases such as diphtheria;pertusis; tetanus; polio, influenza, hepatitis, rotavirus, pneumonia,measles, mumps, rubella, varicella, meningitis, papillomavirus, etc. TherHBHA protein product may also include one or more adjuvants and/or oneor more immunogenicity enhancers, examples of which include but are notlimited to: mineral salts e.g., aluminum hydroxide (“alum”), aluminiumphosphate, calcium phosphate; oil emulsions e.g., MF59, adetergent-stabilised oil-in-water emulsion; particulate adjuvants e.g.,virosomes, ISCOMS (structured complex of saponins and lipids); microbialderivatives e.g., MPL(TM) (monophosphoryl lipid A), CpG motifs, modifiedtoxins, etc; various plant derivatives e.g., saponins (QS-21); as wellas endogenous immunostimulatory adjuvants e.g., cytokines, heat shockproteins (HSPs) and fragments thereof, etc.

The forgoing examples serve to further illustrate particular embodimentsof the invention but should not be interpreted as limiting the inventionin any way.

Examples Example 1. Cloning, Over-Expression and Testing of the HeparinBinding Hemagglutinin (HBHA) in BCG

The purpose of these studies was to develop a recombinant M. bovis BCGstrain over-expressing HBHA A recombinant BCG strain previouslyconstructed at Aeras (AERAS-413) was used because it is a panCDauxotroph and the presence of panCD on the plasmid with the HBHA genewas used for complementation and colony selection in the absence ofantibiotic resistance.

BCG Strain

Initial cloning steps were carried out in Escherichia coli stbl3 cellsgrown in LB supplemented with kanamycin (40 ug/ml). For the overexpression of HBHA, a pantothenate auxotroph of a BCG Danish SSI strainexpressing the pfo gene from Clostridium perfringens (AERAS-413) thatfacilitates antigen presentation through endosome escape mechanisms wasutilized (1) The BCG strain was grown in Middle brook 7H9-broth andsupplemented with glycerol, OADC and D-pantothenic acid (25 ug/ml). Theparent and the recombinant BCG were plated on 7H10-OADC plates with andwithout pantothenate supplement respectively.

Design and CloninK Strategy

The Mtb sequence for HBHA gene was taken from the Tuberculist websiteand was synthesized (DNA 2.0) along with the Ag85B promoter and leaderpeptide in front of the start HBHA codon (FIGS. 1A, 1B, and 2).

The synthesized fragment was cloned between sites PacI and SpeI inpKAMCB2 vector which is an E. coli-mycobacterial shuttle vector with akanamycin resistance marker (aph) and a complementing panCD geneoperated via the hsp60 promoter (FIGS. 3A and 3B). This plasmid was usedso that it could complement the panCD auxotrophy in AERAS-413 and alsomaintain the stability of the plasmid in AERAS-413. Once cloned, theantibiotic marker was digested out with HpaI enzyme and the constructwas self-ligated to make it “antibiotic resistant marker free” (FIG. 4).The self-ligated construct was electroporated into AERAS-413 and therecovered colonies were plated onto 7H10-plates without pantothenatesupplement and without antibiotics. The colonies were screened for thepresence of the antigen, the plasmid backbone and the absence of thekanamycin antibiotic resistance marker.

Amino Acid Comparison of Native and Recombinant HBHA

Native HBHA has 100% sequence identity in both BCG and Mtb. Therecombinant HBHA is expressed using Ag85B promoter and Ag85B leaderpeptide fused to the N-terminus of the protein (FIGS. 5A, 5B, and 5C).

Genotypic Analysis of the Colonies from AERAS-413

The colonies from AERAS-413 plates transformed with the pKAMCB2derivative containing the HBHA gene were screened for the presence ofthe full length Ag85B-HBHA construct in the plasmid. The primers usedfor this PCR were Ag-HBHA.for: GGTCTTCGTCGGCTTGCTTC (SEQ ID NO: 6) andAg-HBHA.rev: GCTCTGCCAGTGTTACAACC (SEQ ID NO: 7) and the product size of2.6 kb was expected which is seen in the colony No. 5 (FIGS. 6A, 6B, and6C). The same colonies were tested for the presence of the plasmidbackbone spanning from the oriM to panCD complementing gene and for theabsence of the antibiotic resistance marker. Primers used for theplasmid backbone were oriM7932.for GTCTACGAGGCCACACTCAG (SEQ ID NO: 8)and pan9969.rev TATCGCGCAGCTCCAGGTAG (SEQ ID NO: 9). Primers used forchecking the kanamycin antibiotic marker m the plasmid werekan_intrnl.for GCTCGAGGCCGCGATTAAATTC (SEQ ID NO: 10) and kan_intrnl.revGGATGGCAAGATCCTGGTATCG (SEQ ID NO: 11). Colony No. 5 shows the presenceof the plasmid backbone and also the absence of the kanamycin gene fromthe backbone making it antibiotic marker less. The recombinant AERAS-413strain over-expressing HBHA was named AERAS-445 and was used for furtherphenotypic analysis and scale-up manufacturing. (FIGS. 6A, 6B, and 6C)

Growth Kinetics of AERAS-445

AERAS-445 grown under conditions described above was observed to have asimilar growth pattern to that of the parent strain AERAS-413 as well asAERAS-401. The seed culture was diluted to the OD₆₀₀ of 0.2 and theabsorbance was measured over 9 days to observe the growth pattern. Thegrowth pattern for AERAS-445 was very similar to that of the parentstrain indicating that the over-expressed HBHA protein did not have anydetrimental effect on the growth kinetics (FIG. 7)

Phenotypic Analysis of the Colonies from AERAS-413

Cultures were grown in protein-free 7H9 media to an OD₆₀₀=1.0. Cultureswere spun at 3,000 rpm for ten minutes to separate the supernatant andpellets. To process the pellets for cell lysates, the pellets wereresuspended in a protease inhibitor cocktail buffer and then treated bybead beating to disrupt the cells. The lysates were centrifuged at3,000×g at 4 degrees to remove debris. Cell lysate proteinconcentrations were measured by BCA (bicinchoninic acid) protein assay.Normalization of samples was performed by loading equal amounts ofprotein, which was thirty micrograms. Samples were prepared by heatingat 70 degrees for fifteen minutes with reducing agent and loading dye.Samples were then loaded onto gradient (4-12% Bis-Tris) polyacrylamidegels and run with MOPS buffer. The transfer was done using the iBlot®Dry blotting system for six minutes. Western blot analysis was doneusing the Snap i.d. system (Millipore). The primary antibody used waseither the anti-HBHA monoclonal 4057D2 (2) (1:2000), or 1G10 (1:1000)anti-HBHA antibody followed by use of goat anti-mouse HRP secondaryantibody (KPL). Detection was done by HRP chemiluminescence (Immun-Star,Biorad).

In FIG. 8, Arrow 1 indicates the recombinant HBHA in AERAS-445 and itsabsence in the control parent strain AERAS-413 and arrow 2 indicates thenative HBHA present in both control AERAS-413 and recombinant AERAS-445.The 4057D2 monoclonal antibody, which recognizes the methylated portionof the HBHA protein, indicated that the recombinant HBHA produced inAERAS-445 is methylated. The Western blot procedure was repeated withthe monoclonal antibody 1G10 developed at the Institut Pasteur de Lille,France. AERAS-401 and AERAS-413 was used as controls in this blot. Arrow1 indicates the presence of recombinant HBHA in AERAS-445 and itsabsence in the control parent strains AERAS-401 and AERAS-413 and arrow2 indicates the native HBHA is present in both the controls andrecombinant strains. Unlike the 4057D2 antibody, 1G10 recognizes aspecific epitope within the non-methylated region of the HBHA protein(FIG. 9).

cGMP Manufacturing of HBHA from the AERAS-445 Strain

Through a gradual reduction in animal-protein concentration, therecombinant BCG (rBCG) was adapted to grow in media free from serum andanimal origin protein. The final product is a concentrated accessioncell bank of 4.5±0.4 mL per vial stored at vapor phase of liquidnitrogen which can be used for the inoculation, e.g. for cGMP MasterCell Bank Production.

In order to adapt the rBCG to growing in a serum and animal originprotein free medium, the use of Oleic Albumin Dextrose Catalase (OADC)supplement in the culture medium during ACB establishment wasprohibited. Table 1 summarizes the process of converting to serum animalorigin protein free medium. The medium used for ACB construction wasModified Middle brook 7H9 Medium (MM7H9) without OADC or any other serumor animal derived supplement. The first stage culture was grown in a 500mL venti-cap flask with no baffles. All cultures after the first stagewere grown in baffled shaker flasks.

TABLE 1 AERAS-445 Growth Guidelines for Accession Cell Bank Flask OADCVol. Vol. Inoc. Total Centrifuge Pass/ Size conc. MM7H9 OADC Vol. Vol.Target At Spec Stage (mL) (%) (mL) (mL) (mL) (mL) A600 (yes/no)? 1 500 095 0 5.0 100 4 ± 1.5 No 2 2000 0 900 0 100 1000 4 ± 1.5 No 3 2000 0 8500 150 125 4 ± 1.5 Yes

After the culture reached its final passage, it was harvested/recovered.The culture was centrifuged at 1200×g for 30 min at 6±4° C. andre-suspended in 10% GST solution stored at room temperature at ¼ theoriginal volume (Table 1). The culture was then dispensed in 4.0±0.5 mLaliquots into 5 mL sterile cryovials. The vials were stored in the vaporphase on liquid nitrogen.

Two vials of post-freeze AERAS-445 were tested for sterility. Theresults were negative for any growth contaminants. Vials containing 4×concentrated AERAS-445 rBCG were frozen at vapor phase liquid nitrogen.As shown in FIG. 10, a Western blot with the anti-HBHA monoclonalantibody 1G10 shows the production of the target antigen (HBHA) fromcell lysates of AERAS-445 frozen cultures.

Procedure for the Development of an Accession Cell Bank of AERAS 445.

-   1. Prepare growth media and freezing media.    -   a. R&D Style Middle brook 7H9

4.7 g/L Middle brook 7H9 Powder 0.24% (v/v) Glycerol 0.05% (w/v)Tyloxapol 10% (v/v) OADC Supplement

-   -   b. Modified Middle brook 7H9

4.7 g/L Middle brook 7H9 Powder 2.0 g/L Sodium Glutamate 2% (v/v)Glycerol 3 mg/L Zinc Sulfate Heptahydrate 0.2 g/L Magnesium SulfateHeptahydrate 0.05% (v/v) Tyloxapol 1.0% (w/v) Dextrose 0% OADCSupplement

-   -   c. 10% GST Solution        -   10% (v/v) Glycerol        -   0.85% (w/v) Sodium Chloride        -   0.05% (v/v) Tyloxapol

-   2. 5 mL of live AERAS-445 culture from Kamal Velmurugan (Vaccine    Discovery) was inoculated into a 500 mL shake flask containing    prewarmed 95 mL culture containing Modified Middle brook 7H9 medium    without OADC.

-   3. For the rest of the process Modified Middle brook 7H9 medium was    used.

-   4. The culture was incubated in a shaker/incubator (Aeras #1230) at    37° C. and 125 rpm.

-   5. The absorbance was measured at 600 nm when culture has a visual    change in turbidity.

-   6. Once culture reached A₆₀₀=4.0±1.5 AU, it was inoculated into the    second stage (900 mL working volume in 2 L shake flask with    venti-cap) with the entire stage 1 culture (˜90 mL).

-   7. The culture was incubated in a shaker/incubator (Aeras #1230) at    37° C. and 125 rpm.

-   8. The absorbance was measured at 600 nm when culture has a visual    change in turbidity.

-   9. Once culture reached A₆₀₀=3.0±1.5 AU, it was inoculated into the    Stage 3 (850 mL MM7H9 medium without OADC) with 150 mL of the Stage    2 culture. Total 3×2 L shake flasks (working volume 1 L/flask) were    inoculated.

-   10. The culture was incubated in a shaker/incubator at 37° C. and    125 rpm.

-   11. The absorbance was measured at 600 nm when culture has a visual    change in turbidity.

-   12. Once the culture (Stage 3) reached A₆₀₀=4±1.5 AU, the flask with    median A₆₀₀ readout was selected and harvesting process was    initiated.

-   13. The selected Stage 3 culture was centrifuged in a pre-autoclaved    1 L centrifuge tube for 30 minutes at 1200×g and 4° C.

-   14. The supernatant was discarded and the pellet was re-suspended in    ¼ volume (˜250 mL) of 10% GST.

-   15. While maintaining mixing, 5 mL cryo-vials were filled with    4.0±0.5 mL of the re-suspended cells.

The filled vials were immediately frozen in vapor phase of liquidnitrogen.

Example 2. Purification and Functional Analysis of rHBHA from AERAS-445Estimation of the Yield of HBHA Protein in AER4S-445

The amount of HBHA produced in AERAS-445 was estimated by a sandwichELISA method developed at the Institut Pasteur de Lille. The primarymonoclonal antibody was 1G10 and the secondary monoclonal antibody wasbiotinylated 5F2, developed at the Institut Pasteur de Lille (EZ-linkSulfo-NHS-LC-Biotin, Pierce). In short, 100 ul per well of mAb 1G10 at 5ug/ml diluted in coating buffer was seeded on to high affinity bindingELISA plate and incubated over night at 4° C. The plate was washed twiceand blocked with PBS-Tween20 (+1% BSA) for 1 hour. Recombinant andnative HBHA were diluted with 1×PBS (8 dilutions) and 100 ul were addedonto each well and incubated for 2 hours. After three washes with1×PBS-Tween20, 100 ul of the mAb 5F2 at a concentration of 0.2 ug/ml wasadded and incubated for 1 hour. The wells were washed thrice and 100 ulStreptavidin-HRP (BD biosciences) conjugate was added at concentrationof 1/1000 in PBS and incubated for 30 mins at room temperature. Theprotein was detected with 100 ul of TMB substrate (ELISA peroxidesubstrate) and developed at room temperature for 5 mins to 15 mins. Thereaction was stopped with 50 ul of 3M H₃PO₄ and the optical density wasread at 450 nm. From this result AERAS-445 expressed almost 8-fold moreHBHA compared to the parent strain (FIGS. 11A and 11B).

The yield of HBHA from the M. bovis BCG Pasteur 1173P2 was also comparedwith AERAS 445. The BCG strains were grown in 200 ml agitated Sautonmedium in 1-liter flasks for 10 days until the optical density readingat 600 nm reached approximately 1.5. The BCG cells were then harvestedby centrifugation at 8,500 rpm (Beckmann J2MC centrifuge, rotor JA 0).The BCG pellet was then suspended in 20 ml PBS+0.05% Tween 80 (Tw 80:Sigma Ultra, ref P8074) and centrifuged at 6,500 rpm (Beckman CoulterAllegra 64R, rotor F0650) for 20 min at 4° C. The pellet was theweighted before suspension in 20 ml PBS+0.05% Tween 80, heat inactivatedand sonicated as described below. The supernatant of the sonicate wasthen analyzed for its protein content and for the concentration of HBHAby using the Sandwich ELISA with the monoclonal antibodies 1G10 and 5F2,as described above. An average of 3 to 4 independent measurementsindicated that BCG Pasteur 1173P2 produces approximately 10 μg HBHA/mgof total protein, whereas AERAS 445 produces an average of approximately19 μg HBHA/mg of total protein. This AERAS 445 produces roughly twice asmuch HBHA as BCG Pasteur 1173P2.

Purification of rHBHA from AERAS 445

Pellets of AERAS 445 were resuspended in 20 ml PBS+0.05% Tween80 (Tw 80,Sigma Ultra, ref. P8074) and centrifuged at 6,500 rpm (Beckman CoulterAllegra 64R, rotor F0650) for 20 min. at 4° C. The pellets were weightedand resuspended in 20 ml PBS+0.05% Tween 80, heated for 30 min. at 80°C. and centrifuged for 20 min. at 12,000 rpm (Beckman Coulter Allegra64R, rotor F0650) at 4° C.

1.5 g of heat-inactivated AERAS 445 was suspended in 15 ml PBS+0.05%Tween80 and sonicated on ice with an Analog sonifier unit model S-450A(Branson, US) for 20 min. (output 7, duty cycle 90) with a flatdisrupter horn (13 mm, VWR ref. 142-3751). The sonicate was centrifugedat 13,500×g for 20 min, and the supernatant was applied onto aheparin-Sepharose CL-B column (1×5 cm) equilibrated with DPBS. Thecolumn was then washed with 100 ml DPBS, and the bound material waseluted by a 0-500 mM NaCl gradient in 100 ml DPBS. The eluted 1-mlfractions were identified by SDS-PAGE using a 12% gel followed byCoomassie Brilliant Blue R-250 staining.

The HBHA-containing fractions were pooled and applied onto areverse-phase HPLC Nucleosil C18 column (TSK gel super ODS; Interchim)equilibrated in 0, 05% trifluoroacetic acid. Bound material was elutedwith a linear 0-80% acetonitrile gradient. HBHA-containing fractionswere pooled, and the solvent was eliminated by evaporation. The pH ofthe final fraction of 1 ml was adjusted to pH8 by using 1 M Tris-HCl(pH9). The final product was stored at −80° C. HBHA-containing fractionswere analyzed by SDS-PAGE, mass spectrometry and sandwich ELISA usingthe 1G10 and the 5F2 monoclonal antibodies, as described above.

FIG. 12 shows a mass-spectrometry analysis of purified rHBHA from AERAS445. For the mass-spectrometry analysis, the protein (0.1 to 10 pmol)was prepared by the dry droplet method. The protein solution (0.5 μl)was mixed with freshly dissolved a-cyano4-hydroxycinnaminic acid at 10mg/ml in 50% CH₃CN and 0.1% trifluoroacetic acid. After spotting anddrying, mass spectrometry analysis was performed by using amatrix-assisted laser desorption ionization/time-of-flight(Voyager-DE-STR, Applied Biosystems). The following setting parameterswere used: positive and linear modes, acceleration voltage of 25 kV,grid voltage of 92%, 750 ns of delayed extraction time, and low massgate of 1,000 Da. The spectra were calibrated externally by using the[M+H+] monoisotopic ions of different peptides for average masses of E.coli thioredoxin and horse apomyoglobin (Applied Riosystems).

Mass Spectrometry Analysis of rHBHA Methylation Pattern

Purified rHBHA was digested overnight with 5% endoproteinaseGlu-C(Roche), and the resulting peptides were separated by reverse-phaseHPLC using a Beckman Ultra sphere ODS column (2×200 mm) and a linear0-60% acetonitrile elution gradient prepared in 0.1% trifluoroaceticacid and directly analyzed by mass spectrometry.

For mass spectrometry, the peptide solution was mixed with freshlydissolved a-cyano4-hydroxycinnaminic acid at 10 mg/ml in 50% CH₃CN and0.1% trifluoroacetic acid. After spotting and drying, mass spectrometryanalysis was performed by using a matrix-assisted laser desorptionionization/time-of-flight Voyager-DE-STR (Applied Biosystems). Forpeptides between 3,000 Da to 10,000 Da, the following setting parameterswere used: positive and reflector modes, acceleration voltage of 25 kV,grid voltage of 65%, 250 ns of delayed extraction, and low mass gate of1,000 Da. The spectra were calibrated externally by using the [M+H+]monoisotopic ions of different peptides for average masses of E. colithioredoxin and horse apomyoglobin (Applied Biosystems).

The results of the mass spectrometry analysis of rHBHA purified fromAERAS 445 are depicted in FIG. 13 and indicate that the C-terminal endof the protein shows heterogeneity, consistent with the complexmethylation profile expected for HBHA.

Reaction of Different Forms of HBHA with Monoclonal Antibodies 4057D2and 3921E4

The concentration of purified rHBHA from AERAS 445 was measured andadjusted in order to coat overnight at 4° C. 1 μg per well of ELISAplates. The plates were then washed with phosphate buffered saline(PBS)/Tween (PBST), followed by blocking for 2 hrs at room temperaturewith PBST containing 3% BSA. After washing again three times with PBST,the plates were incubated for 1 h30 at room temperature with serialfour-fold dilutions of monoclonal antibodies 3921E4 or 4057D2 inPBST+BSA. The latter two preferentially recognize the methylated formsof HBHA (3). The plates were then washed 10 times with PBST andincubated for 1 h at room temperature with anti-mouse antibodies linkedto peroxidase. After 10 additional washings with BPST, 100 μl ofperoxidase substrate TMB was added to each well, and the colour reactionwas stopped by the addition of 50 μl H₃PO₄. Finally the absorbency at450 nm was read in each well using a standard ELISA plate reader. FIGS.14A and 14B show that rHBHA from AERAS 445 is recognized by both the4057D2 and the 3921E4 monoclonal antibodies, confirming that it isproperly methylated.

Antigenicity of rHBHA from AERAS 445 in Latently Infected Humans

Purified rHBHA from AERAS 445 was incubated at 2 g/ml with 10⁶peripheral blood mononuclear cells (PBMC) isolated from three latentlyinfected human subjects, three patients with active tuberculosis andfrom three negative control subjects. After 96-hours, the IFN-γconcentrations released in the supernatants were measured by ELISA, andthe IFN-γ secretion from unstimulated PBMC was subtracted from theantigen-induced IFN-γ secretion. The sensitivity of the IFN-γ (ELISA was10 μg/ml. The cut-off value for optimal discrimination betweennon-infected controls and LTBI subjects was previously determined to be100 pg/ml for HBHA (4). As shown in Table 2, rHBHA purified from AERAS445 is well recognized by the PBMCs from latently infected individuals,poorly recognized by the PBMCs from tuberculosis patients and notrecognized by the PBMCs from healthy controls.

TABLE 2 Human T cell antigenicity of HBHA. The PBMC from 9 humansubjects (3 latently infected subjects Nos. 1, 8 and 9; three activetuberculosis patients Nos. 2, 3 and 4 and 3 negative controls Nos. 5, 6and 7) were incubated with 2 μg/ml of HBHA from M. bovis BCG Pasteur1173P3 (IPL) or AERAS 445 (Aeras). and the resulting IFN-gconcentrations were measured in the culture supernatants and areexpressed as ng/ml HBHAIPL HBHAAeras* Patient Number (BCG Pasteur)(AERAS-445) 1 5.21 13.53 2 0.09 0.02 3 0.02 0.11 4 0.24 0.27 5 0.07 0.126 <0.01 <0.01 7 <0.01 <0.01 8 1.43 .73 9 1.62 1.28

REFERENCES

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While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1-19. (canceled)
 20. A method of determining whether a subject has alatent tuberculosis infection, comprising: detecting the presence orabsence of immune reactivity to a fusion protein comprising an Ag85Bleader sequence attached to an amino terminus of a mycobacterialheparin-binding hemagglutinin (HBHA) protein or antigenic fragmentthereof in said patient, wherein the presence of immune reactivityindicates that said subject has a latent tuberculosis infection.
 21. Themethod of claim 20, wherein said step of detecting comprises: obtaininga biological sample from said subject, and detecting said immunereactivity in said biological sample.
 22. The method of claim 20,wherein said biological sample is a sputum sample or a serum sample. 23.The method of claim 20, wherein said step of detecting comprises:intradermally injecting said subject with said fusion protein, anddetecting immune reactivity at a site of intradermal injection.
 24. Amethod of eliciting an immune response against Mycobacteriumtuberculosis in a subject in need thereof, comprising: administering tosaid subject an amount of a fusion protein comprising an Ag85B leadersequence attached to an amino terminus of a mycobacterialheparin-binding hemagglutinin (HBHA) protein or antigenic fragmentthereof sufficient to elicit an immune response in said subject; oradministering to said subject a Mycobacterium comprising a nucleic acidfusion sequence encoding an Ag85B leader sequence attached to an aminoterminus of a mycobacterial heparin-binding hemagglutinin (HBHA) proteinor antigenic fragment thereof, wherein said fusion sequence is operablylinked to a promoter, and wherein said Mycobacterium is administered inan amount sufficient to elicit an immune response in said subject. 25.The method of claim 24, wherein said immune response is production ofone or more of B cells, antibodies and T cells.
 26. The method of claim24, wherein said immune response is a protective immune response. 27-37.(canceled)
 38. A method of preparing a composition comprising aheparin-binding hemagglutinin (HBHA) protein comprising: growing aculture of Mycobacterium comprising a nucleic acid fusion sequenceencoding an Ag85B leader sequence attached to an amino terminus of amycobacterial heparin-binding hemagglutinin (HBHA) protein or antigenicfragment thereof, wherein said fusion sequence is operably linked to apromoter; obtaining said rHBHA protein from said culture; purifying saidrHBHA protein; and combining said purified rHBHA protein with aphysiologically acceptable carrier.
 39. The method of claim 38, whereinsaid growing is carried out in shake culture or by fermentation.
 40. Themethod of claim 38, further comprising adding at least one agentselected from the group consisting of: one or more antigens that are notHBHA, one or more adjuvants, and one or more immunogenicity enhancers,to said composition. 41-44. (canceled)
 45. The method according to claim20, wherein said antigenic fragment is comprised within the last 30 to50 carboxyl terminal amino acids of SEQ ID NO:4.
 46. The methodaccording to claim 45, wherein said antigenic fragment is or comprisesSEQ ID NO: 5.